WO2022221692A1 - Prophylaxie et thérapie anticancéreuses faisant appel à des nanoparticules virales ciblées - Google Patents

Prophylaxie et thérapie anticancéreuses faisant appel à des nanoparticules virales ciblées Download PDF

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WO2022221692A1
WO2022221692A1 PCT/US2022/025071 US2022025071W WO2022221692A1 WO 2022221692 A1 WO2022221692 A1 WO 2022221692A1 US 2022025071 W US2022025071 W US 2022025071W WO 2022221692 A1 WO2022221692 A1 WO 2022221692A1
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cancer
cpmv
tumor
nanoparticle
virus
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PCT/US2022/025071
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English (en)
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Nicole STEINMETZ
Jooneon Park
Young Hun Chung
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/18011—Comoviridae
    • C12N2770/18023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/18011—Comoviridae
    • C12N2770/18034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/18011—Comoviridae
    • C12N2770/18041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/18043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/18011—Comoviridae
    • C12N2770/18041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/18045—Special targeting system for viral vectors

Definitions

  • Metastatic cancer remains a challenge to treat and diagnose regardless of the cancer’s origin. For instance, the median survival time of breast cancer patients with metastatic recurrences is 2-3 years. 1 Metastatic melanoma tumors are similar in their aggressiveness and prognosis of the disease becomes very difficult once metastasis has been achieved by the primary tumor. 2 Metastasis to the lungs remains one of the most common forms of metastasis in both breast cancer and melanoma.
  • VLP virus or virus like particle
  • the virus or VLP is from a plant virus from the group of the genus Bromovirus, Comovirus, or Tymovirus.
  • the plant virus is selected from Cowpea chlorotic mottle virus (CCMV), Cowpea mosaic virus (CPMV), or Physalis mottle virus (PhMV).
  • the virus or VLP has an exposed lysine side chain.
  • the peptide comprises, or alternatively consists essentially of, or yet further consists of a c-terminal cysteine that can be introduced via a linker.
  • a linker includes the peptide GGGSC.
  • the lysine side chain is conjugated to an N- hydroxysuccinimide (NHS) ester and the maleimide of a maleimide-polyethylene glycol8 is conjugated with the c-terminal cysteine of the peptide.
  • the peptide that recognizes and binds S100A9 comprises or consists of a peptide having the amino acid of one or more of SEQ ID NO: 1 and/or 2, or 1, 2 or 10 to 15 or a immunogenic fragment thereof, or an equivalent of each thereof that is at least 70% identical, or at least 80%, or at least 90% or at least 95%, or at least 98% or similar to one or more of SEQ ID NO: 1 and/or 2, or 1, 2 or 10 to 15 or a immunogenic fragment thereof, respectively, or the immunogenic fragment thereof.
  • SEQ ID NO: 1 and/or 2 equivalent or fragment thereof further comprises, consists essentially of, or consist of a linker with a c-terminal cysteine, e.g., GGGSC.
  • isolated polynucleotides encoding the virus, VLP, nanoparticle of this disclosure or an equivalent thereof.
  • a vector comprising, or alternatively consisting essentially of, or yet further consisting of the virus, VLP, polynucleotide of this disclosure, optionally linked to regulatory or other elements for the expression of the virus, VLP, nanoparticle.
  • the vector genome is expressed from a DNA construct encoding it in a host cell.
  • a host cell comprising, or alternatively consisting essentially of, or yet further consisting of the polynucleotide of this disclosure.
  • a plurality of virus, VLP, or nanoparticles where the virus, VLP, or nanoparticles are the same or different from each other.
  • a composition comprising, or alternatively consisting essentially of, or yet further consisting of the virus, VLP, nanoparticle, polynucleotide, vector and/or host cell.
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of a carrier and one or more of the nanoparticle, polynucleotide, vector and/or the host cell of this disclosure.
  • a method for inducing an immune response comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject one or more of the virus, VLP, nanoparticle, polynucleotide, vector, the composition and/or the host cell of this disclosure.
  • Also provided herein is a method for targeting the tumor microenvironment to reverse immunosuppression of a tumor that optionally secretes S100A the method comprising, or consisting essentially of, or consisting of contacting the tumor microenvironment with the virus, VLP, nanoparticle, polynucleotide, vector, the composition and/or the host cell of this disclosure.
  • the contacting can be in vitro or in vivo.
  • the S100A9 target can be secreted into the tumor microenvironment, but in one aspect, not necessarily expressed on the surface of tumor cells. Targeting the tumor microenvironment will interact and contact immune cells to reverse immunosuppression and launch anti-tumor immunity through activation of the innate and then adaptive immune system.
  • the method can be used to screen for combination therapies or for personalized treatments for animals, mammals and human.
  • the cancer cell is any of the cancer cells described herein, including primary and metastatic cells.
  • the method comprises administering an effective amount of the composition as described herein and can be used to test for new drug combinations (in an animal model) or for therapy itself.
  • the treatment can be combined with other therapies and therefore can be a first line, second line, third line, fourth line or fifth line therapy.
  • Methods to determine effectiveness of the therapy include reduction in tumor size or burden, prolonged progression-free survival, prolonged overall survival and reduced toxicity.
  • One embodiment of the disclosure relates to a method for treating or preventing cancer or metastasis in a subject in need thereof, comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject in need thereof one or more of the virus, VLP, nanoparticle, polynucleotide, vector, the composition and/or the host cell of this disclosure.
  • the cancer is any of the cancer cells described herein, including primary and metastatic cancer.
  • the method comprises administering an effective amount of the composition as described herein.
  • the method can be used to test for new drug combinations or for therapy itself.
  • the treatment can be combined with other therapies and therefore can be a first line, second line, third line, fourth line or fifth line therapy.
  • Methods to determine effectiveness of the therapy include reduction in tumor size or burden, prolonged progression-free survival, prolonged overall survival and reduced toxicity. These methods can be combined with other clinical markers, e.g., a reduction in a tumor marker, e.g., CA125.
  • a method for altering an immune cell profile in lungs of a subject comprising, or alternatively consisting essentially of, or yet further consisting of the virus, VLP, nanoparticle, polynucleotide, vector, the composition and/or the host cell of this disclosure.
  • the method comprises administering an effective amount of the composition as described herein.
  • the method can be used to test for new drug combinations or for therapy itself.
  • the treatment can be combined with other therapies and therefore can be a first line, second line, third line, fourth line or fifth line therapy. Methods to determine effectiveness of the therapy are known in the art and described herein.
  • kits for performing the methods of this disclosure as well as instructions for carrying out the methods of the present disclosure comprises, or alternatively consists essentially of, or yet further consists of one or more of the virus, VLP, nanoparticle, polynucleotide, vector, the composition and/or the host cell of this disclosure
  • the instruction for use provide directions to conduct any of the methods disclosed herein.
  • BRIEF DESCRIPTION OF THE DRAWINGS CPMV bioconjugation strategy. CPMV is first extracted from infected black-eyed pea No. 5 plants. The large and small coat proteins are shown in light and dark grey; surface exposed Lys side chains are highlighted as black spheres.
  • FIGS. 2A – 2E Characterization of CPMV, peptide-conjugated CPMV, and fluorescent CPMV particles.
  • FIG. 2A SDSPAGE of the CPMV particles. The arrows point to H6/G3 peptide-modified coat proteins.
  • FIG. 2B DLS measurements of the CPMV particles. The box in black is displaying the average diameter in nm of the particles (FIG. 2D) and the polydispersity index (PDI).
  • FIG. 2C TEM images of uranyl acetate-stained CPMV particles. Scale bars represent 100 nm.
  • FIG. 2D UV-VIS of the fluorescent Cy5-conjugated CPMV particles.
  • FIGS. 3A – 3E Biodistribution and localization of fluorescent CPMV and CCMV nanoparticles following administration.
  • FIG. 3A Schematic and timeline of the biodistribution study.
  • FIG. 3B IVIS imaging of lungs following CPMV and CCMV nanoparticle injection.
  • FIG. 3B was presented with a color gradient from deep red (10k) to yellow (30K).
  • Lung results have true color for CPMV-G3 for middle and bottom and CPMV-H6 in all three positions
  • FIG. 3C, FIG. 3D Quantitative analysis of the fluorescence signal from the organs after CPMV (FIG. 3C) and CCMV (FIG. 3D) nanoparticle injection.
  • FIG. 3E Confocal imaging indicates colocalization of the CPMV-Cy5-G3 particles with S100A9. Scale bar represents 25 ⁇ m. DAPI staining is shown in blue while S100A9 and CPMV particles are shown in teal and yellow, respectively.
  • FIGS. 4A – 4I CPMV particles show immunoprophylaxis in C57BL/6J mice challenged i.v. with B16F10 melanoma or 4T1-Luc TNBC cells.
  • FIGS. 4A – 4I Schematic and timeline of the B16F10 prophylaxis study.
  • FIG. 4B Harvested lungs were fixed and imaged before manual tumor counting.
  • FIG. 4C Quantitative analysis of the number of tumor nodules found on the surface of the lungs. The middle line indicates the mean number of tumor nodules.
  • FIG. 4D Repeated B16F10 prophylactic immunotherapy study including an H6 peptide only control. The middle line indicates the mean number of tumor nodules.
  • FIG. 4E H&E images of the harvested lungs. The dark purple spots are indicative of the B16F10 tumor nodules in the lungs.
  • FIG. 4F Quantitative analysis of the H&E pictures in (FIG. 4E). The ratio of tumor cells to total cells within the H&E images were plotted. The images were analyzed using QuPath software.
  • FIGS. 5A – 5E S100A9-targeted CPMV immunotherapy against lung metastasis from i.v.
  • FIG. 5A Treatment schedule of the metastatic B16F10 melanoma model using C57BL/6J mice and therapeutic administration of CPMV and CPMV-H6.
  • FIG. 5B Quantitative analysis of tumor nodules counted in lungs harvested post-treatment.
  • FIG. 5C Treatment schedule of the metastatic 4T1-Luc breast cancer model using Balb/c mice.
  • FIG. 5D Quantitative luminescence of the tumors following ROI measurements of the images from (FIG. 5E) Luminescent imaging of the 4T1-luc tumors taken on the IVIS. The mice were imaged every two days following 150 mg/kg i.p.
  • FIG. 6A A RAW-BLUE TM assay comparing the immunogenicity between wild type CPMV, peptide-conjugated CPMV, and the peptide only controls.
  • the CPMV and the peptide- conjugated CPMV particles were strong TLR and/or NOD agonists while the peptides by themselves were not indicating that the peptides themselves do not impart any significant immunotherapeutic effects.
  • FIG. 6B RAWBLUE TM assay comparing the immunogenicity of CPMV to CCMV.
  • CPMV was much more immunogenic compared to CCMV, which helps to explain why in the previous studies, CPMV was able to significantly reduce tumor burden while CCMV did not.
  • FIG. 6C FACS analysis of the immune cell profile following CPMV injection. C57BL/6J mice were i.v. injected with CPMV, CPMV-H6, CPMV-G3, and PBS and the lungs were harvested and analyzed. CPMV-H6 showed greater DC recruitment and DC activation while CPMV-G3 did not recruit more DCs, but did activate them significantly more than controls. CPMV-H6 further recruited neutrophils and macrophages.
  • FIG. 7 CCMV bioconjugation strategy. CCMV is first extracted from infected black-eyed pea No. 5 plants. Surface exposed lysines are highlighted as black spheres.
  • FIGS. 8A – 8E Characterization of CCMV, peptide-conjugated CCMV, and fluorescent CCMV particles.
  • FIG. 8A SDS-PAGE of the CCMV particles. The purple arrow points to H6/G3 peptide-modified coat proteins.
  • FIG. 8B DLS measurements of the CCMV particles.
  • the box in black is displaying the average diameter in nm of the particles (D) and the polydispersity index (PDI).
  • the low PDI indicates minimal aggregation of the CCMV particles following bioconjugation.
  • FIG. 8C TEM images of uranyl acetate-stained CCMV particles. Scale bars represent 100 nm.
  • FIG. 8D UV-VIS of the fluorescent Cy5-conjugated CCMV particles.
  • FIGS 9A – 9D Additional characterization of the non-fluorescent CPMV and CCMV particles.
  • FIG. 9A, FIG. 9B 1.2% (w/v) agarose gel of the CPMV (FIG. 9A) and CCMV (FIG. 9B) particles. Data indicate that particles remain intact and do not aggregate.
  • FIGS. 10A – 10B Additional characterization of dual-tagged peptide and fluorescent CPMV particles.
  • FIGS. 10A – 10B Additional characterization of dual-tagged peptide and fluorescent CPMV particles.
  • FIGS. 10A SDS/PAGE gels of fluorescent CPMV particles.
  • FIGS. 11A – 11C Biodistribution of fluorescent CPMV and CCMV nanoparticles following administration.
  • FIG.11A Schematic and timeline of the biodistribution study.
  • FIGS. 12A – 12B 4T1-Luc immunoprophylaxis study.
  • FIG. 12A Schematic and treatment schedule of CPMV as an immunoprophylaxis in 4T1-Luc metastatic breast cancer.
  • FIG. 12B Luminescent imaging of the 4T1-Luc tumors taken every 3 days. D- luciferin was injected i.p. at a concentration of 150 mg/kg to enable luminescent imaging. Additionally, the mice were weighed and checked for any signs of noticeable weight loss.
  • FIGS. 13A – 13C S100A9-targeted CPMV immunotherapy against lung metastasis from i.v.
  • FIG. 13A Schematic and treatment schedule of the 4T1-Luc breast cancer model using Balb/c mice.
  • FIG. 13B Kaplan-Meier curve of the mice from the 4T1-Luc immunotherapy study. The mice were sacrificed when they reached their clinical endpoints. Wild type CPMV increased the median time of survival by one day while the CPMV-H6 treatment increased the median time of survival by 3 days.
  • FIG. 13C Luminescent imaging of the 4T1-Luc tumors taken on the IVIS. The mice were imaged every two days following 150 mg/kg i.p. injection of D- luciferin, and the luminescence was calculated using ROI measurements from the Living Image 3.0 software.
  • FIG. 14 The gating strategy used in (FIG. 6C) of the flow cytometry analysis of CPMV particles’ ability to stimulate innate immune cells.
  • FIGS 15A – 15C Liver enzyme assays to determine liver toxicity of the CPMV i.v. injections.
  • FIG. 15A Schematic of the ALT and AST assays.
  • FIG. 15B ALT Assay (FIG. 15C) AST assay.
  • FIGS. 16A – 16E Characterization of CPMV, CPMV-H6, and CPMV-G3.
  • FIG. 16A UV-VIS
  • FIG. 16B SDS-PAGE
  • FIGS. 17A – 17B Detection of S100A8/9 within the IP space.
  • FIG. 17A Injection schedule.
  • FIG. 17B ELISA data from IP gavage indicating that S100A8/9 levels within the IP space increase significantly following CT26-Luc (colon cancer) injection.
  • FIGS. 18A – 18D CT26-Luc treatment with S100A9-targeted CPMV particles. Treatment with CPMV-H6/G3 significantly extends survival and slows the growth of the colon cancer.
  • FIG. 18A Injection schedule.
  • FIG. 18B Body weight fold change of mice (FIG.
  • FIGS. 19A – 19C Circumference fold change of mice.
  • FIGS. 19A – 19C Survival curve of mice.
  • FIGS. 19A – 19C Targeting of IP tumors using CPMV-H6/G3.
  • FIG. 19A Injection Schedule.
  • FIG. 19B Quantitative fluorescent measurements of the IP tumors from IVIS imaging. Data indicates a trend for both the CPMV-H6 and CPMV-G3 in targeting the tumors.
  • FIG. 19C Ex vivo imaging of harvested tumors following CT26-Luc IP injection.
  • FIGS. 20A – 20D Additional targeting data.
  • FIG. 20A Lungs
  • FIG. 20B Kidneys
  • FIG. 20C Spleen
  • FIG. 20D Liver.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.
  • compositions or methods include the recited steps or elements, but do not exclude others.
  • Consisting essentially of shall mean rendering the claims open only for the inclusion of steps or elements, which do not materially affect the basic and novel characteristics of the claimed compositions and methods.
  • Consisting of shall mean excluding any element or step not specified in the claim.
  • Embodiments defined by each of these transition terms are within the scope of this disclosure
  • the terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.
  • “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
  • the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term “mammal” includes both human and non-human mammals.
  • subject refers to animals, typically mammalian animals. Any suitable mammal can be treated by a method, cell or composition described herein.
  • mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
  • a mammal is a human.
  • a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a mammal can be a pregnant female.
  • a subject is a human.
  • a subject has or is suspected of having a cancer or neoplastic disorder.
  • “Eukaryotic cells” comprise, or alternatively consist essentially of, or yet further consist of all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton.
  • the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
  • eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human, “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome.
  • Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 ⁇ m in diameter and 10 ⁇ m long).
  • Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral.
  • bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.
  • composition typically intends a combination of the active agent, e.g., the nanoparticle of this disclosure and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • active agent e.g., the nanoparticle of this disclosure and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • Representative amino acid components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose
  • compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
  • unit dose or "dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
  • the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • encode as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • isolated cell generally refers to a cell that is substantially separated from other cells of a tissue. The term includes prokaryotic and eukaryotic cells.
  • immune response or its equivalent “immunological response” refers to the development of a cell-mediated response (e.g. mediated by antigen- specific T cells or their secretion products).
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to treat or prevent a viral infection, expand antigen-specific B-reg cells, TC1, CD4+ T helper cells and/or CD8+ cytotoxic T cells and/or disease generated, autoregulatory T cell and B cell “memory” cells.
  • the response may also involve activation of other components.
  • the term “immune response” may be used to encompass the formation of a regulatory network of immune cells.
  • regulatory network formation may refer to an immune response elicited such that an immune cell, preferably a T cell, more preferably a T regulatory cell, triggers further differentiation of other immune cells, such as but not limited to, B cells or antigen-presenting cells – non-limiting examples of which include dendritic cells, monocytes, and macrophages.
  • regulatory network formation involves B cells being differentiated into regulatory B cells; in certain embodiments, regulatory network formation involves the formation of tolerogenic antigen-presenting cells.
  • immune cells includes, e.g., white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • T cell includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gamma-delta T cells.
  • a “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.
  • Cytokines are small secreted proteins released by immune cells that have a specific effect on the interactions and communications between the immune cells. Cytokines can be pro-inflammatory or anti-inflammatory.
  • Non-limiting example of a cytokine is Granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes.
  • GM-CSF Granulocyte-macrophage colony-stimulating factor
  • vector refers to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc.
  • a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • plasmid vectors may be prepared from commercially available vectors.
  • viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc.
  • the viral vector is a lentiviral vector.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Further details as to modern methods of vectors for use in gene transfer may be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art.
  • an “effective amount” or “efficacious amount” refers to the amount of an agent or combined amounts of two or more agents, that, when administered for the treatment of a mammal or other subject, is sufficient to effect such treatment for the disease.
  • the “effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated. In some embodiments, the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject and the methods in use.
  • the effective amount may comprise, or alternatively consist essentially of, or yet further consist of one or more administrations of a composition depending on the embodiment.
  • the term “disease” or “disorder” as used herein refers to a cancer or a tumor (which are used interchangeably herein), a status of being diagnosed with such disease, a status of being suspect of having such disease, or a status of at high risk of having such disease.
  • cancer or “malignancy” or “tumor” are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize) as well as any of a number of characteristic structural and/or molecular features.
  • a “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include, but not limited to, sarcomas, carcinomas, and lymphomas.
  • a solid tumor comprises bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, esophageal cancer, colon cancer, glioma, cervical cancer, hepatocellular, thyroid cancer, or stomach cancer.
  • a “metastatic cancer” is a cancer that spreads from where it originated to another part of the body.
  • a “cancer cell” are cells that have uncontrolled cell division and form solid tumors or enter the blood stream.
  • administer intends to mean delivery of a substance to a subject such as an animal or human. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and animals, treating veterinarian. Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and the target cell or tissue.
  • route of administration include intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal, and inhalation.
  • An agent of the present disclosure can be administered for therapy by any suitable route of administration.
  • “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response such as passive immunity; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient.
  • a therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
  • the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample.
  • the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.
  • “homology” or “identical”, percent “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding the chimeric PVX described herein).
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • the terms “homology” or “identical,” percent “identity” or “similarity” also refer to, or can be applied to, the complement of a test sequence.
  • the terms also include sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length.
  • An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein.
  • the phrase “first line” or “second line” or “third line” refers to the order of treatment received by a patient.
  • First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively.
  • the National Cancer Institute defines first line therapy as “the first treatment for a disease or condition.
  • primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies.
  • First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008.
  • a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.
  • an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and/or exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.
  • equivalent polypeptide or “equivalent peptide fragment” refers to protein, polynucleotide, or peptide fragment encoded by a polynucleotide that hybridizes to a polynucleotide encoding the exemplified polypeptide or its complement of the polynucleotide encoding the exemplified polypeptide, under high stringency and/or which exhibit similar biological activity in vivo, e.g., approximately 100%, or alternatively, over 90% or alternatively over 85% or alternatively over 70%, as compared to the standard or control biological activity.
  • Additional embodiments within the scope of this disclosure are identified by having more than 60%, or alternatively, more than 65%, or alternatively, more than 70%, or alternatively, more than 75%, or alternatively, more than 80%, or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98% or 99% sequence homology. Percentage homology can be determined by sequence comparison using programs such as BLAST run under appropriate conditions. In one aspect, the program is run under default parameters.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds.1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25 °C to about 37 °C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40 °C to about 50 °C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • a high stringency hybridization refers to a condition in which hybridization of an oligonucleotide to a target sequence comprises no mismatches (or perfect complementarity).
  • high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1x SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • isolated refers to molecules or biologicals or cellular materials being substantially free from other materials.
  • the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
  • protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any aspect of this technology that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • the term “purified” does not require absolute purity; rather, it is intended as a relative term.
  • a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants.
  • substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration.
  • the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients.
  • the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
  • “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • the disease is cancer
  • the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor.
  • treatment excludes prophylaxis.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • the term “overexpress” with respect to a cell, a tissue, or an organ expresses a protein to an amount that is greater than the amount that is produced in a control cell, a control issue, or an organ.
  • a protein that is overexpressed may be endogenous to the host cell or exogenous to the host cell.
  • the term “enhancer”, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed.
  • An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.
  • promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • contacting means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity.
  • Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.
  • the term “introduce” as applied to methods of producing modified cells such as chimeric antigen receptor cells refers to the process whereby a foreign (i.e. extrinsic or extracellular) agent is introduced into a host cell thereby producing a cell comprising the foreign agent.
  • Methods of introducing nucleic acids include but are not limited to transduction, retroviral gene transfer, transfection, electroporation, transformation, viral infection, and other recombinant DNA techniques known in the art.
  • transduction is done via a vector (e.g., a viral vector).
  • transfection is done via a chemical carrier, DNA/liposome complex, or micelle (e.g., Lipofectamine (Invitrogen)).
  • viral infection is done via infecting the cells with a viral particle comprising the polynucleotide of interest (e.g., AAV).
  • introduction further comprises CRISPR mediated gene editing or Transcription activator-like effector nuclease (TALEN) mediated gene editing.
  • TALEN Transcription activator-like effector nuclease
  • Methods of introducing non-nucleic acid foreign agents include but are not limited to culturing the cells in the presence of the foreign agent, contacting the cells with the agent, contacting the cells with a composition comprising the agent and an excipient, and contacting the cells with vesicles or viral particles comprising the agent.
  • culturing refers to growing cells in a culture medium under conditions that favor expansion and proliferation of the cell.
  • culture medium or “medium” is recognized in the art and refers generally to any substance or preparation used for the cultivation of living cells.
  • Media may be solid, liquid, gaseous or a mixture of phases and materials.
  • Media include liquid growth media as well as liquid media that do not sustain cell growth.
  • Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices.
  • Exemplary gaseous media include the gaseous phase to which cells growing on a petri dish or other solid or semisolid support are exposed.
  • the term “medium” also refers to material that is intended for use in a cell culture, even if it has not yet been contacted with cells. In other words, a nutrient rich liquid prepared for culture is a medium.
  • a powder mixture that when mixed with water or other liquid becomes suitable for cell culture may be termed a “powdered medium.”
  • “Defined medium” refers to media that are made of chemically defined (usually purified) components. “Defined media” do not contain poorly characterized biological extracts such as yeast extract and beef broth. “Rich medium” includes media that are designed to support growth of most or all viable forms of a particular species. Rich media often include complex biological extracts.
  • a “medium suitable for growth of a high-density culture” is any medium that allows a cell culture to reach an OD600 of 3 or greater when other conditions (such as temperature and oxygen transfer rate) permit such growth.
  • basal medium refers to a medium which promotes the growth of many types of microorganisms which do not require any special nutrient supplements. Most basal media generally comprise of four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. A basal medium generally serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. In one aspect, the growth medium may be a complex medium with the necessary growth factors to support the growth and expansion of the cells of the disclosure while maintaining their self-renewal capability.
  • basal media examples include, but are not limited to, Eagles Basal Medium, Minimum Essential Medium, Dulbecco’s Modified Eagle’s Medium, Medium 199, Nutrient Mixtures Ham’s F-10 and Ham’s F-12, McCoy’s 5A, Dulbecco’s MEM/F-I 2, RPMI 1640, and Iscove’s Modified Dulbecco’s Medium (IMDM).
  • IMDM Modified Dulbecco’s Medium
  • S100 calcium-binding protein A9 (S100A9; also known as migration inhibitory factor-related protein 14 or MRP14 or calgranulin B) is a protein involved in cellular processes such as cell cycle progression and differentiation and a central mediator of inflammation in cancer and other diseases.8,9 It is a calcium-binding protein that regulates inflammation and while there is some level of endogenous S100A9 expression in the squamous epithelium and mucosal tissues,9,10 it becomes overexpressed in many different forms of cancer including breast, ovarian, skin, bladder, pancreatic, gastric, esophageal, colon, glioma, cervical, hepatocellular, and thyroid.8 , 11–13 It is most commonly found in its heterodimer form with S100A8, but can also be found as a homodimer.12 , 14 , 15 S100A8/9 complexes are also found in mice and extensive biochemical characterization has demonstrated functional equivalency with its human counterpart.16 S100A9
  • H6 MEWSLEKGYTIK SEQ ID NO:1
  • G3 WGWSLSHGYQVK SEQ ID NO: 2
  • VLPs Virus and Virus-like Particles
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system.
  • VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification.
  • Methods for producing VLPs are known in the art.
  • the presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like.
  • VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Viral.
  • the virus or VLP is derived from Cowpea chlorotic mottle virus (CCMV).
  • CCMV Cowpea chlorotic mottle virus
  • CCMV is a spherical plant virus that belongs to the Bromovirus genus.
  • Several strains have been identified and include, but not limited to, Car1 (Ali, et al., 2007. J. Virological Methods 141:84-86), Car2 (Ali, et al., 2007. J. Virological Methods 141:84-86, 2007), type T (Kuhn, 1964.
  • the virus or VLP from CCMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins.
  • the capsid protein is a wild-type CCMV capsid, optionally expressed by Car1, Car2, type T, soybean (S), mild (M), Arkansas (A), bean yellow stipple (BYS), R, or PSM strain.
  • the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and/or deletions.
  • the CCMV capsid comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03601: MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYS VSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSC VTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAALT EGDVIVHLEVEHVRPTFDDSFTPVY (SEQ ID NO: 4), or an equivalent thereof.
  • the virus or VLP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141: 84-86 (2007).
  • the virus or VLP is derived from Cowpea mosaic virus (CPMV).
  • CPMV Cowpea mosaic virus
  • CPMV is a non-enveloped plant virus that belongs to the Comovirus genus.
  • CPMV strains include, but are not limited to, SB (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64:1) and Vu (Agrawal, H.O. (1964). Meded. Landb. Hoogesch.
  • the virus or VLP from CPMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins.
  • CPMV produces a large capsid protein and a small capsid protein precursor (which generates a mature small capsid protein).
  • CPMV capsid is formed from a plurality of large capsid proteins and mature small capsid proteins.
  • the large capsid protein is a wild- type large capsid protein, optionally expressed by SB or Vu strain.
  • the large capsid protein is a modified large capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and/or deletions.
  • the large capsid protein comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833): MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYDVVV NGQDFRATVAFLRTHVITGKIKVTATTNISDNSGCCLMLAINSGVRGKYSTDVYTICS QDSMTWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVIA KLDWSIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGGGAG ATQAFLANMPNSWISMWRYFRGELHFEVTKMSSP
  • the mature small capsid protein is a wild-type mature small capsid protein, optionally expressed by SB or Vu strain.
  • the mature small capsid protein is a modified mature small capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and/or deletions.
  • the mature small capsid protein comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 834-1022): GPVCAEASDVYSPCMIASTPPAPFSDVTAVTFDLINGKITPVGDDNWNTHIYN PPIMNVLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDARTF VISQPGSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNMRFD PNFRVAGNILMPPFPLSTETPPL (SEQ ID NO: 6), or an equivalent thereof.
  • the virus or VLP is derived from Physalis mottle virus (PhMV).
  • PhMV is a single stranded RNA virus that belongs to the genus Tymovirus.
  • the virus or VLP from PhMV comprises, or consists essentially of, or yet further consists of, a plurality of coat proteins.
  • the coat protein is a wild-type PhMV coat protein.
  • the coat protein is a modified coat protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and/or deletions.
  • the PhMV coat comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351: MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSL QTADPITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVY GGQSFVLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTA SIQISGRIRLSKPMLIAN (SEQ ID NO: 7), or an equivalent thereof.
  • the virus or VLP is derived from Sesbania mosaic virus (SeMV).
  • SeMV is a positive stranded RNA virus that belongs to the genus Sobemovirus.
  • the virus or VLP from SeMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins.
  • the capsid protein is a wild-type SeMV capsid protein.
  • the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and/or deletions.
  • the SeMV capsid comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06: MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRSAVQQLQPTQAGISM APSAQGAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWLR GVAANWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGYV SGQVWSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVDV NIATPLVPARLVIALLDGSSSTAVAAGRIYCTYTIQMIEPTASALNN (SEQ ID NO: 9), or an equivalent thereof.
  • a polynucleotide or a protein include a polynucleotide or a protein that comprise, or consists essentially of, or yet further consists of, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identify to the respective polynucleotide or protein of which it is compared to, while still retaining a functional activity.
  • a functional activity refers to the formation of a virus or VLP.
  • a capsid described herein includes, e.g., a modified capsid comprising, or consisting essentially of, or yet further consisting of, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to its respective wild-type version.
  • sequence identity refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • reference sequence refers to a molecule to which a test sequence is compared.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to a reference sequence means that, when aligned, that percentage of bases (or amino acids) at each position in the test sequence are identical to the base (or amino acid) at the same position in the reference sequence.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment.
  • One alignment program is BLAST, using default parameters.
  • the term “conservative substitution” denotes the replacement of an amino acid residue by another, chemically or biologically similar residue.
  • Biologically similar means that the substitution does not destroy a biological activity or function, e.g., assembly of a viral capsid.
  • ⁇ Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size.
  • Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic.
  • conservative substitutions include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the substitution of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • the term "conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.
  • proteins that include amino acid substitutions can be encoded by a nucleic acid. Consequently, nucleic acid sequences encoding proteins that include amino acid substitutions are also provided.
  • Modified proteins also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms.
  • Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.
  • Modified forms further include “chemical derivatives,” in which one or more amino acids has a side chain chemically altered or derivatized.
  • Such derivatized polypeptides include, for example, amino acids in which free amino groups form amine hydrochlorides, p- toluene sulfonyl groups, carobenzoxy groups; the free carboxy groups form salts, methyl and ethyl esters; free hydroxl groups that form O-acyl or O-alkyl derivatives as well as naturally occurring amino acid derivatives, for example, 4-hydroxyproline, for proline, 5- hydroxylysine for lysine, homoserine for serine, ornithine for lysine etc. Also included are amino acid derivatives that can alter covalent bonding, for example, the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide.
  • a virus or VLP described herein further comprise, or consists essentially of, or yet further consists of, a label or a tag, e.g., such as a detectable label.
  • a detectable label can be attached to, e.g., to the surface of a virus or VLP.
  • Non-limiting exemplary detectable labels also include a radioactive material, such as a radioisotope, a metal or a metal oxide. Radioisotopes include radionuclides emitting alpha, beta or gamma radiation.
  • a radioisotope can be one or more of: 3 H, 10 B, 18 F, 11 C, 14 C, 13 N, 18 O, 15 O, 32 P, P 33 , 35 S, 35 Cl, 45 Ti, 46 Sc, 47 Sc, 51 Cr, 52 Fe, 59 Fe, .57 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As 76 Br, 77 Br, 81m Kr, 82 Rb, 85 Sr, 89 Sr, 86 Y, 90 Y, 95 Nb, 94m Tc, 99m Tc, 97 Ru, 103 Ru, 105 Rh, 109 Cd, 111 In, 113 Sn, 113m In, 114 In, I 125 , I 131 , 140 La, 141 Ce, 149 Pm, 153 Gd, 157 Gd, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Er, 169 Y, 1
  • a metal or metal oxide is one or more of: gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodynium, indium, or technetium.
  • a metal oxide includes one or more of: Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Ffe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), or Er(III).
  • detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); magnetic and paramagnetic agents (e.g., iron-oxide chelate); nanoparticles; an enzyme (horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase); a prosthetic group (e.g., streptavidin/biotin and avidin/biotin); a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); a luminescent material (e.g., luminol); or a bioluminescent material (e.g., lumino
  • tags and/or detectable labels include enzymes (horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (e.g., biotin); receptors (avidin); GST-, T7-, His-, myc-, HA- and FLAG®-tags; electron-dense reagents; energy transfer molecules; paramagnetic labels; fluorophores (fluorescein, fluorscamine, rhodamine, phycoerthrin, phycocyanin, allophycocyanin); chromophores; chemi-luminescent (imidazole, luciferase, acridinium, oxalate); and bio-luminescent agents.
  • enzymes horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-gal
  • a detectable label or tag can be linked or conjugated (e.g., covalently) to the virus or VLP or nanoparticle.
  • a detectable label such as a radionuclide or metal or metal oxide can be bound or conjugated to the agent, either directly or indirectly.
  • a linker or an intermediary functional group can be used to link the molecule to a detectable label or tag.
  • Linkers include amino acid or peptidomimetic sequences inserted between the molecule and a label or tag so that the two entities maintain, at least in part, a distinct function or activity.
  • Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character which could promote or interact with either domain.
  • Amino acids typically found in flexible protein regions include Gly, Asn and Ser.
  • the length of the linker sequence may vary without significantly affecting a function or activity.
  • Linkers further include chemical moieties, conjugating agents, and intermediary functional groups. Examples include moieties that react with free or semi-free amines, oxygen, sulfur, hydroxy or carboxy groups.
  • Such functional groups therefore include mono and bifunctional crosslinkers, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo- SMPB), in particular, disuccinimidyl suberate (DSS), BS3 (Sulfo-DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST).
  • sulfo-SMCC sulfo-SMCC, sulfo- SMPB
  • DSS disuccinimidyl suberate
  • BS3 Sulfo-DSS
  • DSG disuccinimidyl glutarate
  • DST disuccinimidyl tartrate
  • Non-limiting examples include diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid.
  • virus, VLP, or nanoparticle as described herein further comprising, or consisting essentially of, or yet further consisting of the peptide that recognizes and binds S100A9 and an additional therapeutic agent.
  • additional therapeutic agent disclosed herein comprise, or consists essentially of, or yet further consists of, a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof.
  • Illustrative additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5-fluorouracil (5-FU), 6- mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors such as topotecan or irinotecan (CPT- 11); topoisomerase II inhibitors such as etoposide (VP- 16), teni
  • the virus, VLP, or nanoparticle with or without the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, or is used as a first-line therapy.
  • first-line therapy comprises, or consists essentially of, or yet further consists of, a primary treatment for a subject with a cancer.
  • the cancer is a primary cancer.
  • the cancer is a metastatic or recurrent cancer.
  • the first-line therapy comprise, or consists essentially of, or yet further consists of, chemotherapy.
  • the first-line treatment comprise, or consists essentially of, or yet further consists of, radiation therapy.
  • first-line treatments may be applicable to different type of cancers.
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, or is used as a second-line therapy, a third-line therapy, a fourth-line therapy, or a fifth-line therapy.
  • a second-line therapy encompasses treatments that are utilized after the primary or first-line treatment stops. They can also be used as third- line, fourth-line or fifth line therapy.
  • a third-line therapy, a fourth-line therapy, or a fifth-line therapy encompass subsequent treatments.
  • a third- line therapy encompass a treatment course upon which a primary and second-line therapy have stopped.
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a salvage therapy. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a palliative therapy.
  • the treatment can comprise an additional therapeutic agent that comprises, or consists essentially of, or yet further consists of, an inhibitor of the enzyme poly ADP ribose polymerase (PARP).
  • PARP poly ADP ribose polymerase
  • Exemplary PARP inhibitors include, but are not limited to, olaparib (AZD-2281, LYNPARZA®, from Astra Zeneca), rucaparib (PF- 01367338, RUBRACA®, from Clovis Oncology), niraparib (MK-4827, ZEJULA®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from Abb Vie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).
  • olaparib AZD-2281, LYNPARZA®, from Astra Zeneca
  • rucaparib PF- 01367338, RUBRACA®, from Clovis Oncology
  • niraparib MK-48
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an immune checkpoint inhibitor.
  • exemplary checkpoint inhibitors include: PD-L1 inhibitors such as Genentech' s MPDL3280A (RG7446), anti-PD-Ll monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol -Meyer's Squibb, MSB0010718C, and AstraZeneca's MEDI4736; PD-L2 inhibitors such as GlaxoSmithKline's AMP -224 (Amplimmune), and rHIgM12B7; PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMP1-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck's MK-3475 anti-mouse PD-1
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, pembrolizumab, nivolumab, tremelimumab, or ipilimumab. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a cytokine.
  • cytokines include, but are not limited to, IL- ⁇ , IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNF ⁇ .
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a receptor agonist.
  • the receptor agonist comprise, or consists essentially of, or yet further consists of, a Toll-like receptor (TLR) ligand.
  • TLR ligand comprise, or consists essentially of, or yet further consists of, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • the TLR ligand comprise, or consists essentially of, or yet further consists of, a synthetic ligand such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib- OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2p, CFA, or Flagellin.
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an adoptive T cell transfer (ACT) therapy.
  • ACT adoptive T cell transfer
  • ACT involves identification of autologous T lymphocytes in a subject with, e.g., anti-tumor activity, expansion of the autologous T lymphocytes in vitro, and subsequent reinfusion of the expanded T lymphocytes into the subject.
  • ACT comprise, or consists essentially of, or yet further consists of, use of allogeneic T lymphocytes with, e.g., anti-tumor activity, expansion of the T lymphocytes in vitro, and subsequent infusion of the expanded allogeneic T lymphocytes into a subject in need thereof.
  • the additional therapeutic agent is, or can be used as a vaccine, optionally, an oncolytic virus.
  • Exemplary oncolytic viruses include T-Vec (Amgen), G47A (Todo et al.), JX-594 (Sillajen), CG0070 (Cold Genesys), and Reolysin (Oncolytics Biotech).
  • the virus or VLP or nanoparticle formulation described herein is administered in combination with a radiation therapy.
  • Modes for Carrying Out the Disclosure Nanoparticles Applicant demonstrates herein that prophylaxis and treatment of lung metastasis could be achieved by targeting immunostimulatory nanoparticles to the lung, which specifically target S100A9 (otherwise known as myeloid-related protein 14).
  • S100A9 is a calcium- binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche as well as post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. S100A9- specific peptide ligands were selected and presented on immunostimulatory nanoparticles derived from cowpea mosaic virus (CPMV) and CCMV.
  • CPMV cowpea mosaic virus
  • a nanoparticle comprises, or alternatively consists essentially of, or yet further consists of a virus or VLP such as CPMV or CCMV and a S100A9 (otherwise known as myeloid-related protein 14) targeting peptide.
  • the S100A9 targeting peptide comprises, or alternatively consisting essentially of, or yet further consisting a peptide selected from H6 peptide or G3 peptide.
  • the virus, VLP, or nanoparticle comprises the amino acid of one or more of SEQ ID NO: 1 and/or 2, or 1, 2 or 10 to 15 an immunogenic fragment thereof, or an equivalent thereof at least 70% identical or similar to one or more of SEQ ID NO: 1 and/or 2, or 1, 2 or 10 to 15 or the immunogenic fragment thereof.
  • the S100A9 targeting peptide further comprises a linker, that optionally contains a c-terminal cysteine, e.g., GGGSC.
  • the virus or VLP (e.g., CMPV or CCMV) has an exposed lysine side chain.
  • the nanoparticle, virus or VLP and/or targeting peptide can be detectably labeled for diagnostic or research purposes.
  • Non-limiting exemplary detectable labels also include a radioactive material, such as a radioisotope, a metal or a metal oxide. Radioisotopes include radionuclides emitting alpha, beta or gamma radiation.
  • a radioisotope can be one or more of: 3 H, 10 B, 18 F, 11 C, 14 C, 13 N, 18 O, 15 O, 32 P, P 33 , 35 S, 35 Cl, 45 Ti, 46 Sc, 47 Sc, 51 Cr, 52 Fe, 59 Fe, .57 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 72 As 76 Br, 77 Br, 81m Kr, 82 Rb, 85 Sr, 89 Sr, 86 Y, 90 Y, 95 Nb, 94m Tc, 99m Tc, 97 Ru, 103 Ru, 105 Rh, 109 Cd, 111 In, 113 Sn, 113m In, 114 In, I 125 , I 131 , 140 La, 141 Ce, 149 Pm, 153 Gd, 157 Gd, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Er, 169 Y, 1
  • a metal or metal oxide is one or more of: gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodynium, indium, or technetium.
  • a metal oxide includes one or more of: Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Ffe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), or Er(III).
  • detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); magnetic and paramagnetic agents (e.g., iron-oxide chelate); nanoparticles; an enzyme (horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase); a prosthetic group (e.g., streptavidin/biotin and avidin/biotin); a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); a luminescent material (e.g., luminol); or a bioluminescent material (e.g., lumino
  • tags and/or detectable labels include enzymes (horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (e.g., biotin); receptors (avidin); GST-, T7-, His-, myc-, HA- and FLAG®-tags; electron-dense reagents; energy transfer molecules; paramagnetic labels; fluorophores (fluorescein, fluorscamine, rhodamine, phycoerthrin, phycocyanin, allophycocyanin); chromophores; chemi-luminescent (imidazole, luciferase, acridinium, oxalate); and bio-luminescent agents.
  • enzymes horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-gal
  • virus or VLP as described herein further comprising, or consisting essentially of, or yet further consisting of the peptide that recognizes and binds S100A9 and an additional therapeutic agent.
  • additional therapeutic agent disclosed herein comprise, or consists essentially of, or yet further consists of, a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof.
  • Illustrative additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors such as topotecan or irinotecan (CPT-11); topoisomerase II inhibitors such as etoposide (VP- 16), teniposide
  • the additional therapeutic can be conjugated to the virus or VLP using methods known in the art and as described herein.
  • the treatment can comprise an additional therapeutic agent that comprises, or consists essentially of, or yet further consists of, an inhibitor of the enzyme poly ADP ribose polymerase (PARP).
  • PARP poly ADP ribose polymerase
  • Exemplary PARP inhibitors include, but are not limited to, olaparib (AZD-2281, LYNPARZA®, from Astra Zeneca), rucaparib (PF- 01367338, RUBRACA®, from Clovis Oncology), niraparib (MK-4827, ZEJULA®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from Abb Vie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).
  • olaparib AZD-2281, LYNPARZA®, from Astra Zeneca
  • rucaparib PF- 01367338, RUBRACA®, from Clovis Oncology
  • niraparib MK-48
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an immune checkpoint inhibitor.
  • exemplary checkpoint inhibitors include: PD-L1 inhibitors such as Genentech' s MPDL3280A (RG7446), anti-PD-Ll monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol -Meyer's Squibb, MSB0010718C, and AstraZeneca's MEDI4736; PD-L2 inhibitors such as GlaxoSmithKline's AMP -224 (Amplimmune), and rHIgM12B7; PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMP1-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck's MK-3475 anti-mouse PD-1
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, pembrolizumab, nivolumab, tremelimumab, or ipilimumab. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a cytokine.
  • cytokines include, but are not limited to, IL- ⁇ , IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNF ⁇ .
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a receptor agonist.
  • the receptor agonist comprise, or consists essentially of, or yet further consists of, a Toll-like receptor (TLR) ligand.
  • TLR ligand comprise, or consists essentially of, or yet further consists of, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • the TLR ligand comprise, or consists essentially of, or yet further consists of, a synthetic ligand such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib- OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2p, CFA, or Flagellin.
  • the additional therapeutic agent is, or can be used as a vaccine, optionally, an oncolytic virus.
  • Exemplary oncolytic viruses include T-Vec (Amgen), G47A (Todo et al.), JX-594 (Sillajen), CG0070 (Cold Genesys), and Reolysin (Oncolytics Biotech).
  • the peptide can be chemically conjugated or genetically fused to the CMPV or CCMV. Any bioconjugation or chemical conjugation method would be applicable for the conjugation.
  • Non-limiting examples of chemical conjugation include conjugating a thiol-terminated peptide through a maleimide-PEG-NHS linker targeting lysine groups on the virus or VLP, e.g., CMPV or CCMV.
  • a lysine side chain is conjugated to a N-hydroxysuccinimide (NHS) ester and the maleimide of a maleimide-polyethylene glycol8 is conjugated with the c-terminal cysteine of the targeting peptide.
  • NHS N-hydroxysuccinimide
  • Azide/alkyne modified peptides and virus or VLP (CMPV or CCMV) and click chemistry can also be used for chemical conjugation.
  • the peptide is added as N-terminal fusion in a CMPV or CCMV plasmid containing the entire VLP (e.g., CMPV or CCMV genome).
  • the diameter of the nanoparticle disclosed herein is from about 10nm to 50nm. In some embodiments, the diameter may range from about 10nm, about 15nm, about 20nm, about 25nm, about 30nm, about 35nm, about 40nm, about 45nm, to about 50nm.
  • a polynucleotide encodes a nanoparticle as disclosed herein that can include regulatory elements, promoters, enhancer and the like, for expression and/or replication.
  • a vector as disclosed herein comprises, or alternatively consists essentially of, or yet further consists of a nanoparticle as disclosed herein.
  • a host cell that comprises, or alternatively consists essentially of, or yet further consists of a virus, VLP, nanoparticle, vector or polynucleotide as disclosed herein.
  • the vector is a plasmid.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is a plant cell or a bacterium.
  • compositions in another aspect, provided herein is a composition comprising, consisting essentially of, or consisting of the combination of formulations comprising a virus, VLP, nanoparticle, polynucleotide, or host cell as provided herein, and at least one carrier, such as a pharmaceutically acceptable carrier or excipient.
  • the composition further comprises a preservative or stabilizer.
  • this technology relates to a composition comprising a combination of nanoparticles or formulations as described herein and a carrier.
  • this technology relates to a pharmaceutical composition comprising a combination of virus, VLP, nanoparticles or formulations as described herein and a pharmaceutically acceptable carrier.
  • this technology relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount or a therapeutically effective amount of a combination of virus, VLP, nanoparticle formulations as described herein and a pharmaceutically acceptable carrier.
  • Compositions including pharmaceutical compositions comprising, consisting essentially of, or consisting of the nanoparticle formulation alone or in combination of other therapeutic agents can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. These can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the combinations of compounds provided herein into preparations which can be used pharmaceutically.
  • the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal administration routes.
  • parenteral administration comprise, or consists essentially of, or yet further consists of, intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra- articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration.
  • the pharmaceutical composition is formulated for local administration. In other instances, the pharmaceutical composition is formulated for systemic administration.
  • the pharmaceutical formulations include, but are not limited to, lyophilized formulations, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form.
  • Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like.
  • Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like.
  • the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids
  • bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane
  • buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
  • the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range
  • Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions
  • suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, sodium citrate, ammonium sulfate and/or other agents such as heparin to increase the solubility and in vivo stability of polypeptides.
  • the pharmaceutical formulations further include diluent which are used to stabilize compounds because they can provide a more stable environment.
  • Salts dissolved in buffered solutions are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.
  • diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling.
  • Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as AVICEL ® , dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di- PAC ® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.
  • the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance.
  • disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or AMIJEL ® , or sodium starch glycolate such as PROMOGEL ® or EXPLOTAB ® , a cellulose such as a wood product, methylcrystalline cellulose, e.g., AVICEL ® , AVICEL ® PH101, AVICEL ® PH102, AVICEL ® PH105, ELCEMA ® P100, EMCOCEL ® , VIVACEL ® , MING TIA ® , and SOLKA-FLOC ® , methylcellulose, croscarmellose,
  • the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials.
  • Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (STEROTEX ® ), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, STEAROWET ® , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CARBOWAXTM, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as SYLOIDTM, CAB-O-SIL ® ,
  • Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.
  • Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N- methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.
  • Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.
  • Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, poly sorb ate-20 or TWEEN® 20, or trometamol.
  • Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g
  • Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., PLURONIC ® (BASF), and the like.
  • compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., PLURONIC ® (BASF), and the like.
  • BASF PLURONIC ®
  • Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes.
  • Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.
  • Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.
  • the pharmaceutical compositions for the administration of the combinations of compounds can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy.
  • compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • each compound of the combination provided herein is included in an amount sufficient to produce the desired therapeutic effect.
  • pharmaceutical compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation.
  • the combination of compounds can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art.
  • Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.
  • Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles.
  • the compositions may also contain formulating agents, such as suspending, stabilizing, and/or dispersing agents.
  • the formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.
  • the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use.
  • a suitable vehicle including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use.
  • the combination of compounds provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
  • the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc, or silica
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); binding agents (e.g. starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc).
  • the tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan.
  • the pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions. Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore TM , or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.
  • one or more compositions disclosed herein are contained in a kit.
  • kits comprising, consisting essentially of, or consisting of one or more compositions disclosed herein and instructions for their use.
  • Dosage and Dosage Formulations the compositions are administered to a subject suffering from a condition as disclosed herein, such as a human, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.
  • Administration of the virus, VLP, VLPs or nanoparticle formulation alone or in combination with the additional therapeutic agent and compositions containing same can be effected by any method that enables delivery to the site of action.
  • intraduodenal routes include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration.
  • Bolus doses can be used, or infusions over a period of 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120 or more minutes, or any intermediate time period can also be used, as can infusions lasting 3, 4, 5, 6, 7, 8, 9, 10, 12, 1416, 20, 24 or more hours or lasting for 1-7 days or more.
  • Infusions can be administered by drip, continuous infusion, infusion pump, metering pump, depot formulation, or any other suitable means. Dosage regimens can be adjusted to provide the optimum desired response.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient can also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient.
  • dosage values can vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values.
  • one or more of the methods described herein further comprise, or consists essentially of, or yet further consists of, a diagnostic step.
  • a sample is first obtained from a subject suspected of having a disease or condition described above.
  • Exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord
  • the sample is a tumor biopsy.
  • the sample is a liquid sample, e.g., a blood sample.
  • the sample is a cell-free DNA sample.
  • Various methods known in the art can be utilized to determine the presence of a disease or condition described herein or to determine whether an immune response has been induced in a subject. Assessment of one or more biomarkers associated with a disease or condition, or for characterizing whether an immune response has been induced, can be performed by any appropriate method. Expression levels or abundance can be determined by direct measurement of expression at the protein or mRNA level, for example by microarray analysis, quantitative PCR analysis, or RNA sequencing analysis.
  • compositions of the present disclosure can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), oral, by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant
  • oral by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel
  • Therapeutic Methods Further disclosed herein are methods for inducing an immune response in a subject consisting essentially of, or yet further consisting of the virus, VLP or nanoparticles, polynucleotides, vectors and/or host cells as disclosed herein. Also provided are methods for targeting the tumor microenvironment to reverse immunosuppression of a tumor that optionally secretes S100A, the methods comprising, or consisting essentially of, or consisting of contacting the tumor microenvironment with the virus, VLP, nanoparticle, polynucleotide, vector, the composition and/or the host cell of this disclosure. The contacting can be in vitro or in vivo.
  • the S100A9 target can be secreted into the tumor microenvironment, but in one aspect, not necessarily expressed on the surface of tumor cells. Targeting the tumor microenvironment will interact and contact immune cells to reverse immunosuppression and launch anti-tumor immunity through activation of the innate and then adaptive immune system. Further disclosed herein are methods for treating cancer in a subject in need thereof, comprising, or alternatively consisting essentially of, or yet further consisting of administering to the subject the virus, VLP, nanoparticles, polynucleotides, vectors and/or host cells as disclosed herein.
  • a subject is a mammal.
  • a subject is a human.
  • a subject has a condition.
  • a subject has cancer.
  • a cancer is selected from melanoma, breast cancer, prostate cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, pancreatic cancer, gastric cancer, esophageal cancer, colon cancer, glioma, cervical cancer, hepatocellular cancer, or thyroid cancer.
  • the cancer is primary or metastatic cancer.
  • the cancer is metastatic or primary lung cancer or breast cancer.
  • the cancer is a primary or metastatic cancer in lung.
  • the cancer expresses or secretes S100A9.
  • administering is selected from intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal, or inhalation.
  • administering is intravenous.
  • the methods and compositions disclosed herein may further comprise or alternatively consist essentially of, or yet further consists of administering to the subject an anti-tumor therapy other than the virus, VLP, nanoparticle disclosed herein.
  • anti- tumor therapy may include different cancer therapy or tumor resection.
  • the additional therapeutic can be combined in the same composition or separately administered.
  • the nanoparticle and/or composition are provided to prevent the symptoms of cancer from occurring in a subject that is predisposed or does not yet display symptoms of the cancer.
  • the virus, VLP, polynucleotide, nanoparticle, vector, or composition disclosed herein may be delivered or administered into a cavity formed by the resection of tumor tissue (i.e. intracavity delivery) or directly into a tumor prior to resection (i.e. intratumoral delivery).
  • the administering is intravenous.
  • any of the virus, VLP, polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a day.
  • any of polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times a week.
  • any of the polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 times a month.
  • any of the virus, VLP, polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • any of the virus, VLP, polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks.
  • any of the virus, VLP, polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, any of the virus, VLP, polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks.
  • any of the virus, VLP, polynucleotides, nanoparticles, vectors, or compositions disclosed herein are administered to the subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months.
  • the methods or compositions further comprise administration of an additional therapeutic agent.
  • the additional therapeutic agent disclosed herein comprise, or consists essentially of, or yet further consists of, a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof.
  • additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idar
  • the virus, VLP, nanoparticle with or without the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, or is used as a first-line therapy.
  • first-line therapy comprises, or consists essentially of, or yet further consists of, a primary treatment for a subject with a cancer.
  • the cancer is a primary cancer.
  • the cancer is a metastatic or recurrent cancer.
  • the first-line therapy comprise, or consists essentially of, or yet further consists of, chemotherapy.
  • the first-line treatment comprise, or consists essentially of, or yet further consists of, radiation therapy.
  • first-line treatments may be applicable to different type of cancers.
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, or is used as a second-line therapy, a third-line therapy, a fourth-line therapy, or a fifth-line therapy.
  • a second-line therapy encompasses treatments that are utilized after the primary or first-line treatment stops. They can also be used as third- line, fourth-line or fifth line therapy.
  • a third-line therapy, a fourth-line therapy, or a fifth-line therapy encompass subsequent treatments.
  • a third- line therapy encompass a treatment course upon which a primary and second-line therapy have stopped.
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a salvage therapy. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a palliative therapy.
  • the treatment can comprise an additional therapeutic agent that comprises, or consists essentially of, or yet further consists of, an inhibitor of the enzyme poly ADP ribose polymerase (PARP).
  • PARP poly ADP ribose polymerase
  • Exemplary PARP inhibitors include, but are not limited to, olaparib (AZD-2281, LYNPARZA®, from Astra Zeneca), rucaparib (PF- 01367338, RUBRACA®, from Clovis Oncology), niraparib (MK-4827, ZEJULA®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from Abb Vie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).
  • olaparib AZD-2281, LYNPARZA®, from Astra Zeneca
  • rucaparib PF- 01367338, RUBRACA®, from Clovis Oncology
  • niraparib MK-48
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an immune checkpoint inhibitor.
  • exemplary checkpoint inhibitors include: PD-L1 inhibitors such as Genentech' s MPDL3280A (RG7446), anti-PD-Ll monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol -Meyer's Squibb, MSB0010718C, and AstraZeneca's MEDI4736; PD-L2 inhibitors such as GlaxoSmithKline's AMP -224 (Amplimmune), and rHIgM12B7; PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMP1-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck's MK-3475 anti-mouse PD-1
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, pembrolizumab, nivolumab, tremelimumab, or ipilimumab. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab. In some cases, the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a cytokine.
  • cytokines include, but are not limited to, IL- ⁇ , IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNF ⁇ .
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, a receptor agonist.
  • the receptor agonist comprise, or consists essentially of, or yet further consists of, a Toll-like receptor (TLR) ligand.
  • TLR ligand comprise, or consists essentially of, or yet further consists of, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • the TLR ligand comprise, or consists essentially of, or yet further consists of, a synthetic ligand such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib- OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2p, CFA, or Flagellin.
  • the additional therapeutic agent comprise, or consists essentially of, or yet further consists of, an adoptive T cell transfer (ACT) therapy.
  • ACT adoptive T cell transfer
  • ACT involves identification of autologous T lymphocytes in a subject with, e.g., anti-tumor activity, expansion of the autologous T lymphocytes in vitro, and subsequent reinfusion of the expanded T lymphocytes into the subject.
  • ACT comprise, or consists essentially of, or yet further consists of, use of allogeneic T lymphocytes with, e.g., anti-tumor activity, expansion of the T lymphocytes in vitro, and subsequent infusion of the expanded allogeneic T lymphocytes into a subject in need thereof.
  • the additional therapeutic agent is, or can be used as a vaccine, optionally, an oncolytic virus.
  • kits for performing the methods of this disclosure as well as instructions for carrying out the methods of the present disclosure.
  • the kit comprises, or alternatively consists essentially of, or yet further consists of one or more of virus, VLP, nanoparticle, polynucleotide, vector and/or host cell of this disclosure and instructions for use.
  • kits are useful for detecting the presence of cancer such as lung cancer in a biological sample e.g., any bodily fluid including, but not limited to, e.g., sputum, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or blood and including biopsy samples of body tissue.
  • a biological sample e.g., any bodily fluid including, but not limited to, e.g., sputum, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or blood and including biopsy samples of body tissue.
  • the test samples may also be a tumor cell, a normal cell adjacent to a tumor, a normal cell corresponding to the tumor tissue type, a blood cell, a peripheral blood lymphocyte, or combinations thereof.
  • test sample used in the above- described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.
  • Methods for preparing protein extracts or membrane extracts of cells are known in the art and can be readily adapted in order to obtain a sample which is compatible with the system utilized.
  • the kit components e.g., reagents
  • the kit can also comprise, or alternatively consist essentially of, or yet further consist of, e.g., a buffering agent, a preservative or a protein-stabilizing agent.
  • the kit can further comprise, or alternatively consist essentially of, or yet further consist of components necessary for detecting the detectable-label, e.g., an enzyme or a substrate.
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art.
  • kit components may be provided in solution or as a liquid dispersion or the like.
  • HBSS Hank’s Balanced Salt Solution
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • PBS phosphate buffered saline
  • FBS Fetal bovine serum
  • BSA Bovine serum albumin
  • Penicillin/streptomycin, potassium phosphate monobasic and dibasic anhydrous powders, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) buffer, sodium acetate anhydrous (NaOAc), methanol, glacial acetic acid, and Sulfo-Cyanine5 (Cy5)-N-hydroxysuccinimide (NHS) esters were purchased from Thermo Fisher Scientific.
  • DMSO dimethyl sulfoxide
  • S(PEG) 8 maleimide- polyethylene glycol8-succinimidyl ester
  • sucrose 10% (v/v) neutral-buffered formalin solution
  • EDTA ethylenediaminetetraacetic acid
  • Bouin Bouin’s solution
  • D-luciferin potassium salt was purchased from Gold Biotechnologies.
  • Ethanol (EtOH) was purchased from VWR International.
  • Paraformaldehyde was purchased from Electron Microscopy Sciences.
  • Mouse 4T1-Luc CL-2539-LUC2
  • B16F10 CL-6475 cells were purchased from ATCC.
  • 4T1-Luc and B16F10 cells were passaged and grown in RPMI-1640 and DMEM respectively and supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin. The cells were incubated at 37°C in a 5% CO2 chamber.
  • RAW- Blue TM Cells (Invivogen, San Diego, CA) were maintained in selection media containing Zeocin (Invivogen) and Normocin (Invivogen) as per instructions by the supplier.
  • CPMV and CCMV nanoparticles were propagated in black eyed pea plants and purified as reported in previous work. 35,67 CPMV was kept at 10 mM potassium phosphate (KP) buffer (pH 7.0-7.2) to the concentration of 2 mg/mL while CCMV was kept in 10 mM NaOAc and 1mM EDTA at pH 4.8 (from here on out called Buffer B). SM(PEG) 8 (5 equivalents) dissolved in DMSO was added to the CPMV particle solution and mixed at room temperature (RT) for 2 hours.
  • KP potassium phosphate
  • Buffer B Buffer B
  • the solution was ultracentrifuged at 4°C at 52000 g for 1 hour with a 40% sucrose cushion. The resulting pellet was resuspended in 10 mM KP, and 0.5 equivalents of H6 (MEWSLEKGYTIKGGGSC) or G3 (WGWSLSHGYQVKGGGSC) peptides were added and mixed at RT for 2 hours. 22 The solution was then dialyzed using a porous membrane tubing (12-14kDa, Spectrum Labs) at RT overnight in 10 mM KP to remove unconjugated peptides. CCMV nanoparticles were diluted to 2 mg/mL in 0.1 M HEPES buffer (pH 7.2).
  • SM(PEG)8 (5 equivalents) was added and allowed to incubate at RT for 2 hours.
  • the buffer was exchanged to Buffer B using a 10 kDa molecular weight cut off (MWCO) filter, and the resuspended pellet was allowed to sit at RT for 2 hours.
  • the solution was ultracentrifuged at 52000 g for 1 hour, resuspended in Buffer B, and diluted with 0.1 M HEPES. A half equivalent of the corresponding peptide (H6 or G3) was then added and mixed at RT for 2 hours.
  • the buffer was exchanged once more and pelleted with ultracentrifugation as before. The final pellet was resuspended in Buffer B.
  • CPMV particles were diluted to 4 mg/mL and an equal number of equivalents of sulfo-Cy5-NHS esters and SM(PEG) 8 were added. The particles were incubated for 2 hours at RT shielded from light and centrifuged using a 100 kDa MWCO filter for 10-12 minutes at 14000 g. The pellet was resuspended with 5 mM KP buffer and H6 and G3 peptides were added (0.5 equivalents) to the solution. The solution was mixed at RT for 2 hours on an orbital shaker.
  • the solution was then dialyzed using a 12-14 kD MWCO molecular porous membrane tubing (Spectrum Labs) at RT overnight in 10 mM KP.
  • CCMV fluorescent particles were diluted to 2 mg/mL in 0.1M HEPES (pH 7.2) buffer. Equal number of equivalents of SM(PEG)8 and sulfo-Cy5-NHS esters were added, and the mixture was incubated at RT away from light for 2 hours.
  • the buffer was then exchanged to Buffer B using a 10 kDa MWCO filter and kept in Buffer B for 2 hours at RT before pelleting down through ultracentrifugation at 52000 g for 1 hour.
  • the particles were resuspended in Buffer B and diluted with 0.1M HEPES before adding the H6 and G3 peptides (0.5 equivalents). The resulting solution was mixed at RT away from light for 2 hours. The buffer was exchanged once more and pelleted with ultracentrifugation as before. The final pellet was resuspended in Buffer B.
  • Particle Characterization Denaturing gel-electrophoresis (SDS-PAGE) CPMV and CCMV samples were diluted in 100 mM KP or 10 mM Buffer B, respectively, and loaded with 4 ⁇ LDS Sample Buffer (Life Technologies) for a final concentration of 10 ⁇ g in 24 ⁇ L.
  • the particles were then denatured at 95°C for 5 minutes and loaded onto a 12% NuPAGE gel (ThermoFisher Scientific) and ran at 200 V, 120 mA, and 25 W for 40 minutes in 1 ⁇ morpholinepropanesulfonic acid (MOPS) buffer (ThermoFisher Scientific).
  • the gels were first destained in a mixture of deionized (DI) water, methanol, and acetic acid (50:40:10; v/v) for 30 minutes followed by staining in 0.25% (wt/vol) Coommassie Blue solution for 30 minutes before imaging with the AlphaImager system (Protein Simple).
  • CPMV particles were diluted in 100 mM KP; CCMV particles were diluted in 10 mM Buffer B. 6 ⁇ Gel Loading Purple dye (Biolabs) was added to the CPMV samples. Instead of lithium dodecyl sulfate, glycerol (3 ⁇ L) was added to CCMV, and 5 ⁇ g of the virus particles were loaded onto a 0.8% (w/v; for CPMV) or 1 % (w/v; for CCMV) agarose gel. With CCMV, the gels were run at 4°C.
  • the agarose gel was stained with 1 ⁇ L of GelRed Nucleic Acid Gel Stain (Gold Biotechnologies) and run for 30 minutes at 120 V and 400 mA. Immediately after the run, the gel was imaged using the AlphaImager system (Protein Simple) under UV light and then imaged again after staining with 0.25% (wt/vol) Coomassie Blue.
  • Dynamic Light Scattering (DLS) A Zetasizer Nano ZSP/Zen5600 (Malvern Panalytical) was used for DLS measurements, and the CPMV and CCMV particles were diluted to 0.5 mg/mL in 10mM KP and 10 mM Buffer B respectively. The particles were run at 25°C with 3 measurements per sample.
  • FPLC Fast protein liquid chromatography
  • CPMV and CCMV were diluted to 0.1 mg/mL in 10 mM KP buffer or 10 mM Buffer B and run through a Superose 6 size-exclusion column at 0.5 mL/min for a total volume of 50 mL in an ⁇ KTA Explorer FPLC machine (GE Healthcare LifeSciences).
  • the elution profile was isocratic, and the UV detectors were fixed at 260 (nucleic acid) and 280 nm (protein).
  • UV-VIS Ultraviolet-Visible Spectroscopy
  • UV-VIS UV-VIS
  • TM concentration of VNPs in the solutions.
  • the fluorescent CPMV and CCMV particles were diluted in 0.1 mM KP and 10 mM Buffer B respectively and measured at 260, 280, and 647 nm to calculate the number of conjugated Cy5 particles per VNP. Concentration of the VNP solutions were carried out using the 260 nm wavelength readings.
  • CPMV, CCMV, and the Cy5 dye are 8.1 mL mg -1 cm -1 , 5.85 mL mg -1 cm -1 , and 270,000 cm -1 M -1 , respectively.
  • Transmission Electron Microscopy (TEM) The CPMV and CCMV samples were imaged using a FEI Tecnai Spirit G2 BioTWIN TEM. The samples were loaded onto Formvar carbon film coated TEM supports with 400- mesh hexagonal copper grids (VWR International) at concentrations ranging from 0.25-1 mg/mL in DI H 2 O for 2 min. The grids were washed with DI H 2 O twice for 45 seconds and then stained with 2% uranyl acetate (Agar Scientific) for 30 seconds twice.
  • the lungs were harvested and then imaged and quantified for fluorescence using the IVIS (Xenogen).
  • Further confocal imaging Nekon A1R Confocal/TIRF STORM microscope
  • B16F10-inoculated mice lungs was accomplished using CPMV-Cy5-PEG and CPMV-Cy5- G3 particles (20 mg/kg).
  • mice were sacrificed and briefly perfused with 10 ml of PBS.
  • the harvested lungs were embedded in OCT medium (Fisher Healthcare) and frozen using liquid nitrogen. They were sliced into 10 ⁇ m thick sections and mounted on microscope glass slides for immunofluorescence staining. OCT residue was removed using PBS.
  • the tissue sections were blocked with 10% (w/v) bovine serum albumin (BSA) in PBS for 1 h and washed with PBS. Staining was accomplished with ⁇ -S100A9 (1:100 dilution, R&D systems, AF2065) and fluorescently-labeled secondary PE ⁇ -goat IgG (1:20 dilution) antibodies prepared in 1% (v/v) BSA.
  • BSA bovine serum albumin
  • mice were imaged with the IVIS using luminescent imaging every 3 days by injecting intraperitoneally (i.p.) 150 mg/kg of body weight D-luciferin. ROI measurements were taken through the Living Image 3.0 software. The weight of the mice was also tracked every 3 days. After 25 days (20 days after tumor inoculation), the lungs were collected and fixed in Bouin’s solution for 3 days. Each tumor nodule on the lungs was counted manually and averaged between the mice in each group.
  • Treatment of B16F10 Melanoma and 4T1 Metastasis Using CPMV Particles B16F10 cells (40,000/mouse) were injected i.v. into female C57B6/J mice (n 7-12).
  • Immunogenicity Profile of CPMV and CCMV Particles in vitro The immunogenicity of CPMV and CCMV was compared through a RAW-Blue TM assay (Invivogen).
  • RAW-Blue TM cells/well were incubated with 0.5 ⁇ g of CPMV and CCMV, 50 EU mL-1 E.coli endotoxin standard control (ThermoScientific), or culture media for 18 hours.
  • Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) stimulation was assessed by measuring the levels of secreted embryonic alkaline phosphatase (SEAP) using a QUANTI-Blue TM (Invivogen) assay. Absorbance was measured at 655 nm using a Tecan microplate reader. The immunogenicity between CPMV and its peptide-conjugated counterparts was also assessed using a RAW-Blue TM assay.
  • RAW-Blue TM cells were incubated with 10 ⁇ g of CPMV, CPMV-H6, CPMV-G3, lipopolysaccharide (LPS), and H6 and G3 peptide for 24 hours.
  • a QUANTI-Blue TM assay was run like before, and absorbance was measured at 655 nm.
  • the protocol is as follows: 50 ⁇ L of antibody solution was added to each well in a 96 well plate, followed by the addition of 100 ⁇ L of cell suspension. Cells were incubated at RT for 30 min in the dark. After 30 min, cells were centrifuged at 500 g for 5 min, the supernatant was aspirated, and the plate was vortexed to loosen cells. Cells were washed by adding 300 ⁇ L of FACS solution and repeating centrifugation. After staining, cells were fixed using 2% (w/v) paraformaldehyde (PFA) in PBS for 15 min, washed once, and resuspend in 200 ⁇ L FACS solution. The cells were stored at 4°C overnight prior to measurement.
  • PFA paraformaldehyde
  • BD FACScelesta (BD Bioscience) and FlowJo were used for data acquisition and analysis, respectively.
  • the blood was spun down at 7,500 rpm for 10 min at 4°C and the sera was collected and stored at -80°C.
  • the sera were then subjected to AST and ALT activity testing by following the manufacturer’s guidelines (Abcam). Briefly, the sera were diluted 10 ⁇ , and compared against a standard curve of pyruvate and glutamate for the ALT and AST assay, respectively. Fluorometric readings at 535 nm (excitation) and 587 nm (emission) and at 10 and 40 min were used to measure ALT activity while AST activity was measured using absorbance readings at 450 nm also at 10 and 40 min (Tecan plate reader). Statistical Analysis All figures and data analysis were created and accomplished using Prism 5 (GraphPad Software).
  • the capsid of CPMV particles consist of 60 copies each of a large and small CP (42 and 24 kDa, respectively) while CCMV particles consist of 180 copies of one 20 kDa CP.
  • SDS-PAGE confirmed successful conjugation with higher molecular weight bands detectable for the CPs.
  • the molecular weight of the peptides are 1846 and 1809 g/mol for the H6 and G3 peptides, respectively; therefore, the band pattern is consistent with a mosaic of unmodified and peptide- displaying CPs for both the CPMV and CCMV formulations (FIG. 1 and FIG. 7).
  • FIG. 9C DLS measurements were consistent with the reported size of CPMV and CCMV 29,35 and indicate presence of monodisperse nanoparticles with hydrodynamic diameters of approximately 30 nm (FIG. 2B and FIG. 8B).
  • the low polydispersity indices (FIG. 2B and FIG. 8B black box) indicate none to minimal aggregation of the particles after peptide conjugation.
  • the G3-conjugated CPMV and CCMV particles did showcase some level of aggregation although this was not deemed largely significant to warrant exclusion from future studies. Differences in the charge of G3, which is net positive at neutral pH, and H6, which is net neutral, may explain the aggregation of CPMV-G3 and CCMV-G3.
  • UV-VIS measurements and the Beer Lambert law were used to determine the number of dyes per particles, and we found consistent labeling with ⁇ 50 Cy5 labels conjugated to CPMV, CPMV-H6, and CPMV-G3 (FIG. 2D).
  • ⁇ 50 Cy5 labels conjugated to CPMV, CPMV-H6, and CPMV-G3 For CCMV, ⁇ 35 Cy5 labels were conjugated to CCMV, CCMV-H6, and CCMV-G3 (FIG. 8D).
  • FPLC analysis was consistent with intact and labeled VNPs being eluted from the column (FIG. 2E and FIG. 8E).
  • the dye co-elutes (absorbance measured at 647) with the RNA and protein signals (measured as 260 nm and 280 nm, respectively) indicating successful conjugation.
  • the absorbance ratio at 260/280 nm is consistent with intact particles; the absorbance ratio of intact CPMV and CCMV is 1.8.
  • Biodistribution of CPMV and CCMV particles Both healthy and B16F10 tumor-bearing mice were injected with native and H6/G3- conjugated CPMV and CCMV; to enable tracking the nanoparticles were labeled with Cy5. Lungs and other organs were harvested 24 h following intravenous (i.v.) nanoparticle injection and imaged ex vivo to observe localization of the nanoparticles in the lungs, spleen, kidney, and liver (FIG.3A).
  • CPMV particles showed no association with S100A9 (not shown).
  • CPMV-Cy5-G3 particles strongly co- localized with S100A9 as indicated by fluorescent overlaps between the CPMV-Cy5-G3 (shown in yellow) and the S100A9 (shown in teal) (FIG. 3E).
  • C57BL/6J mice were pre-exposed to the CPMV (therapeutic) and CCMV (control) nanoparticles with and without the H6/G3 targeting ligands, one week before being challenged i.v. with B16F10 melanoma cells (FIG. 4A).
  • Lungs were harvested 2 weeks post tumor challenge, and tumor nodules were manually counted.
  • the repeated experiment produced very similar results with the CPMV-H6 and CPMV-G3 particles s featuring significantly reduced tumor nodules compared to the PBS (5.6 (p ⁇ 0.0001) and 3.2 fold (p ⁇ 0.0001), respectively) and H6 peptide only controls (5.2 (p ⁇ 0.0001) and 3.0 fold (p ⁇ 0.0001), respectively).
  • the CPMV-H6 formulations had a 5-fold enhanced efficacy vs.
  • CPMV 0.077
  • the lungs of the CPMV mice were further examined through histology and H&E staining (FIG. 4E).
  • the histology slides exemplify that the CPMV treatment greatly reduces tumor burden.
  • tumor cells dark purple
  • the ratio of tumor cells to total cells was analyzed using QuPath software, which illustrates that the CPMV-H6 and CPMV-G3 treatment quite significantly reduces tumor cell count.
  • CPMV-H6 and CPMV-G3 reduced the ratio of tumor:total cells by 18- fold (p ⁇ 0.0001) and 5-fold (p ⁇ 0.0001), respectively.
  • native CPMV displayed efficacy yet at significantly lower levels achieving only 1.7-fold reduction (p ⁇ 0.0001) (FIG. 4F). 4T1-Luc i.v.
  • mice To determine whether the prophylactic effect of S100A9-targeted CPMV could be replicated in other tumor models, we first exposed Balb/C mice to CPMV and S100A9- targeted CPMV by i.v. injection, and then challenged mice with luciferase-labeled 4T1 (4T1- Luc) cells.
  • This experimental lung metastatic model mimics metastatic triple negative breast cancer (TNBC). Tumor cell challenge was carried out 5 days post CPMV exposure (FIG. 4G).
  • Bioluminescent imaging was carried out using the in vivo imaging system (IVIS) imager to track 4T1-Luc cells, and imaging of the PBS treated animals shows that lung metastases established within 2 weeks post tumor cell challenge.
  • IVIS in vivo imaging system
  • FIG. 12B The weight of the mice in both CPMV groups stayed fairly consistent throughout the experiment without any significant loss; no apparent side effects were observed (FIG. 12B).
  • the lungs were harvested after 25 days and fixed in Bouin’s solution before manual counting of tumor nodules (FIG. 4H, FIG. 4I).
  • the CPMV particles were additionally tested as a potential immunotherapy and administered in both B16F10 and 4T1-Luc inoculated mice after establishment of the disease.
  • the CPMV only control did not show any significant decrease in tumor nodules compared to the PBS only control.
  • the wild type CPMV and the peptide-conjugated CPMV exhibited higher level of activation of transcriptional factors (i.e., NF- ⁇ B and AP-1) compared to CCMV control nanoparticles.
  • the H6/G3 peptides alone were not immunostimulatory indicating, as expected, the peptides alone are not TLR and NOD agonists.
  • the immunogenicity assays confirm that CPMV acts as an immunostimulatory adjuvant.
  • lungs were collected 24 h post CPMV treatment and innate immune cell profiles were analyzed using flow cytometry.
  • CPMV-G3 did not significantly improve DC recruitment although it did increase neutrophil infiltration by 1.4 (p ⁇ 0.0001) and 1.3 fold (p ⁇ 0.0001) compared to PBS and native CPMV, respectively.
  • CPMV-H6 additionally increased macrophage infiltration by 1.7 (p ⁇ 0.0001) and 1.5 fold (p ⁇ 0.0001) compared to PBS and CPMV, respectively, although this effect was not observed with CPMV- G3.
  • CPMV-G3 and CPMV-H6 performed equally with insignificant differences between the two for DC activation and M1 macrophage polarization.
  • CPMV cowpox mosaic virus
  • S100A9 is expressed in lung metastasis as well as in many tumor types. Examples include but are not limited to including ovarian, skin, bladder, pancreatic, gastric, esophageal, colon, glioma, cervical, hepatocellular, and thyroid cancer. S100A9 is also expressed in a wide range of cell types.
  • Examples include but are not limited to granulocytes, monocytes, osteoclasts, early myeloid lineage cells, platelets, and cancer cells. It can be expressed, secreted, or displayed, and secretion can be active or passive (i.e. neutrophil necrosis). These factors make S100A9 an attractive target and supports various tumor treatments to be targeted to S100A9. Further supporting this approach is the fact that S100A9 is secreted and found throughout the tumor microenvironment, making it an attractive target to direct nanoparticles and immunotherapies to tumors and metastatic disease. This disclosure relates to composition and treatments that recognize and exploit these new findings. Applicant demonstrate herein that i.v.
  • S100A9-targeted CPMV homes to the lungs and that the CPMV nanoparticle adjuvant effectively immunomodulates the lung environment to recruit DCs and neutrophils while polarizing macrophages to the M1 phenotype protecting mice from i.v. challenge with melanoma and TNBC.
  • the S100A9- targeted CPMV also was effective to treat lung metastasis from melanoma or TNBC after establishment of the disease.
  • Targeted immunotherapies, such as the S100A9-targeted CPMV can be a powerful treatment paradigm to treat high-risk patients and prevent metastatic outgrowth.
  • the standard of care for metastatic cancer is chemotherapy, but this often fails due to development of resistance and/or necessary dose reduction due to harsh side effects.
  • the CPMV nanoparticles are recognized by innate immune cells and signal through pattern recognition receptors leading to release of immunostimulatory cytokines including interleukin (IL)-1 ⁇ , IL-12, interferon (IFN)- ⁇ chemokine ligand 3 (CCL3), macrophage inflammatory protein (MIP)-2, and granulocyte- macrophage colony-stimulating factor (GM-CSF) leading to monocyte recruitment.
  • IL interleukin
  • IFN interferon
  • MIP macrophage inflammatory protein
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • multivalent display of S100A9-targeting ligands directs the CPMV nanoparticles to the lung TME and induces treatment as evident by reduced tumor burden in the lungs after mice were i.v. challenged using melanoma cells or TNBCs (FIG.4).
  • S100A9 was used to target CPMV to the lungs because lung metastases are one of the most common sites of metastasis and prognosis is poor. 51 In both men and women, the lungs were the third highest site of metastasis while in specific cancers such as genital cancers, metastatic growth to the lungs was the most common. Once metastasis occurs, survival rates are low and novel therapies to extend survival must be continuously researched and implemented.
  • S100A9 is also expressed in a wide range of cell types including granulocytes, monocytes, osteoclasts, early myeloid lineage cells, platelets, and cancer cells. 13,52 It can be expressed, secreted, or displayed, and secretion can be active or passive (i.e. neutrophil necrosis).
  • Characterization of the S100A9- targeted nanoparticles of CPMV demonstrated stable formulation chemistry, as PLC, and TEM which indicate the lack of substantial aggregation and structural uniformity of the viruses regardless of conjugation (FIG. 2 and FIG. 8).
  • Denatured gels indicate a mosaic of conjugated and unconjugated coat proteins with up to 24 and 31 peptides per CPMV and CCMV nanoparticle, respectively. This equates to roughly 20 and 17% coat protein conjugation.
  • the facile conjugation scheme producing monodisperse and highly conjugated viral nanoparticles is a key determinant in advancing the translatability and scalability of the CPMV platform.
  • H6/G3 peptides to direct cargo to the TME was previously demonstrated when H6 and G3 peptides were conjugated to the Fc region of mouse IgG2b antibodies to specifically target S100A9 and deplete MDSCs within the TME. 22 Previous work has also explored using small molecule drugs and neutralizing antibodies to block S100A9 function. 8,21,22 However, to the best of Applicant’s knowledge, S100A9 has never previously been targeted in immunotherapy. The exact functional role of S100A9 in cancer and tumorigenesis is not entirely understood, but the protein acts upon immune and tumor cells to modulate the TME into an immunosuppressive state thereby promoting tumor progression and aggressiveness.
  • the CPMV particle without targeting showed some degrees of efficacy.
  • the improved efficacy of the S100A9-targeted formulations can be attributed to tissue targeting resulting in modulation of the lung microenvironment where tumor metastasis occurs.
  • the targeted CPMV was similarly able to improve clinical outcomes in both the melanoma and breast cancer studies (FIG. 5).
  • CPMV-H6 administration decreased B16F10 tumor nodules by 2.7-fold compared to PBS (p ⁇ 0.0001) and 2.3-fold compared to native CPMV (FIG. 5B).
  • CPMV immune cell recruitment and activation
  • DCs, neutrophils, and macrophages were recruited to the lungs by administration of CPMV-H6; DCs were increased by 40-50% (p ⁇ 0.0001), neutrophils by 70-80% (p ⁇ 0.0001), and macrophages by 50-70% (p ⁇ 0.0001) compared to controls.
  • CPMV-G3 did not significantly improve immune cell recruitment; however, it did increase the number of active DCs by 12.1 fold (p ⁇ 0.0001) and polarized 10.8 fold (p ⁇ 0.0001) more M1 tumor-killing macrophages compared to PBS.
  • M1 macrophages are potent tumor cell killers and have tumor-homing properties.
  • 59 You et al. have shown that the number of M1 populations within the tumor islets in non-small cell lung cancer was positively correlated with patient survival.
  • 60 DC activation can reduce immunosuppressive DC states and decrease tumorigenesis through the cross-priming of cytotoxic T-cells and the release of immunostimulatory cytokines such as IL-12, IFN- ⁇ , and Fms-related tyrosine kinase 3 (FLT3).
  • 61,62 Together, data indicate that CPMV is a versatile cancer immunotherapy and its use could be extended beyond localized in situ treatments. As with other nanoparticle-based therapeutics i.v.
  • CPMV prime-boost administration schedules can be established or slow-release could be programmed through applications of long-lasting formulations with microneedles, polymers, scaffolds, or metal-organic frameworks.
  • S100A9 has been recognized as a targetable protein with high expression in multiple tumor types.
  • S100A9-targeted nanoparticles from CPMV home to the lungs When administered prior to tumor challenge, CPMV treatment acts as a prophylaxis and local immunomodulation (recruitment of DCs, neutrophils, and polarization of M1 macrophages) prevents lungs metastasis.
  • the treatment was also effective when administered after disease establishment. This therapy can be a powerful prophylactic approach for high-risk patients, i.e.
  • CT26-Luc cells were maintained in RPMI supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. FBS and penicillin/streptomycin were purchased from Cytiva. The cells were maintained at 37 ⁇ C at 5% CO 2 levels. Production of S100A9-Targeted Cowpea Mosaic Virus Nanoparticles Cowpea mosaic virus (CPMV) was isolated from infected black eyed pea plants as done previously [70] . CPMV was stored in 100 mM potassium phosphate (KP) buffer pH 7.2 at 4 ⁇ C until later use.
  • KP potassium phosphate
  • the H6 (MEWSLEKGYTIKGGGSC) and G3 (WGWSLSHGYQVKGGGSC) peptides were purchased from GenScript Biotech (San Diego), stored in DMSO, and conjugated to the CPMV virus nanoparticles (VNPs) as previously reported [71] .
  • an SMPEG8 linker diluted in DMSO was added to CPMV at 5 equivalents per coat protein (CP) and incubated at room temperature (RT) for 2 h. The excess linker was removed through ultracentrifugation at 52000 g for 1 h with a 40% (w/v) sucrose cushion.
  • the absorbance values at 260 and 280 nm were measured, and the concentration of CPMV within the sample was calculated using the 260 nm wavelength and Beer’s Law with an extinction coefficient of 8.1 mL mg -1 cm -1 .
  • the ratio of 260 to 280 nm (260280 -1 of ⁇ 1.8) was used to determine the absence of broken particles and protein contaminants within the samples.
  • the gels were run at 200 V, 120 mA, and 25 W for 40 min in 1x MOPS buffer, and the protein stains were visualized using GelCode TM Blue Stain reagent (ThermoFisher Scientific) according to the manufacturer’s instructions. The gels were then imaged under an AlphaImager System (Protein Simple). Agarose gel electrophoresis The WT and S100A9-targeted CPMV NPs were diluted to a final concentration of 10 ⁇ g in 30 ⁇ L and run through a 1.2% (w/v) agarose gel stained with GelRed nucleic acid gel stain (Gold Biotechnology). The parameters for running the agarose gel were set at 120 V and 400 mA for 30 min.
  • RNA within the CPMV was then imaged using the AlphaImager System, and the gel was then stained overnight in 0.25% (w/v) Coomassie Blue followed by destaining in a solution of deionized water, methanol, and acetic acid in a 50:40:10 (v:v:v) ratio.
  • the resulting protein bands were imaged using the AlphaImager System.
  • Dynamic light scattering (DLS) The size of the nanoparticles was measured using a Zetasizer Nano ZSP/Zen5600 (Malvern Panalytical) system. The samples were diluted to 0.1 mg mL -1 in 10 mM KP and run at RT.
  • FPLC Fast protein liquid chromatography
  • IVIS in vivo imaging system
  • the IP fluid was then analyzed using a mouse S100A8/9 DuoSet ELISA detection kit (R&D Systems) according to the manufacturer’s instructions.
  • An S100A8/9 kit was utilized as opposed to a monomeric S100A9 kit as S100A9 is mainly found within the body in its heterodimer form with S100A8 [72] .
  • Colon Cancer Treatment Using S100A9-Targeted CPMV The efficacy of S100A9-targeted CPMV was compared to WT CPMV and controls in an IP model of CT26-Luc. BALB/C mice at 6-7 weeks old were injected IP with 500000 CT26-Luc cells in 200 ⁇ L of PBS.
  • the amount of H6 or G3 peptide injected was determined through densitometry analysis of the SDS-PAGE gels (ImageJ).
  • the mice were re-injected at weeks 2 and 3 for a total of 3 injections. The circumference and body weight of the mice were measured every two days starting from the fourth day following tumor cell injection, and the survival of the mice was also followed.
  • ROI region of interest
  • Neoantigens in cancer immunotherapy Science 348, 69–74 (2015). 42. Castle, J. C., Uduman, M., Pabla, S., Stein, R. B. & Buell, J. S. Mutation-Derived Neoantigens for Cancer Immunotherapy. Front Immunol 10, 1856 (2019). 43. Fares, C. M., Van Allen, E. M., Drake, C. G., Allison, J. P. & Hu-Lieskovan, S. Mechanisms of Resistance to Immune Checkpoint Blockade: Why Does Checkpoint Inhibitor Immunotherapy Not Work for All Patients? Am Soc Clin Oncol Educ Book 39, 147–164 (2019). 44. Wang, C., Beiss, V.
  • Heterologous Prime-Boost Enhances the Antitumor Immune Response Elicited by Plant-Virus-Based Cancer Vaccine. J Am Chem Soc 141, 6509–6518 (2019). 50. Lizotte, P. H. et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nat Nanotechnol 11, 295–303 (2016). 51. Riihimäki, M., Thomsen, H., Sundquist, K., Sundquist, J. & Hemminki, K. Clinical landscape of cancer metastases. Cancer Med 7, 5534–5542 (2018). 52. Ghoneum, A., Afify, H., Salih, Z., Kelly, M.

Abstract

L'invention concerne des méthodes et des compositions pour le traitement du cancer. Les compositions et les méthodes contiennent une nanoparticule et un peptide ciblant S100A9.
PCT/US2022/025071 2021-04-16 2022-04-15 Prophylaxie et thérapie anticancéreuses faisant appel à des nanoparticules virales ciblées WO2022221692A1 (fr)

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WO2023034229A1 (fr) * 2021-08-30 2023-03-09 The Regents Of The University Of California Point de contrôle immunitaire ciblant des nanoparticules thérapeutiques
CN116421738A (zh) * 2023-02-28 2023-07-14 中国医科大学附属第一医院 捕获抗原的溶瘤病毒及其药物组合与制备和应用
CN117205152A (zh) * 2023-02-23 2023-12-12 南京大学 一种药物载体，其制备方法及其在疾病治疗中的应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023034229A1 (fr) * 2021-08-30 2023-03-09 The Regents Of The University Of California Point de contrôle immunitaire ciblant des nanoparticules thérapeutiques
CN117205152A (zh) * 2023-02-23 2023-12-12 南京大学 一种药物载体，其制备方法及其在疾病治疗中的应用
CN117205152B (zh) * 2023-02-23 2024-04-09 南京大学 一种药物载体，其制备方法及其在疾病治疗中的应用
CN116421738A (zh) * 2023-02-28 2023-07-14 中国医科大学附属第一医院 捕获抗原的溶瘤病毒及其药物组合与制备和应用
CN116421738B (zh) * 2023-02-28 2024-03-19 中国医科大学附属第一医院 捕获抗原的溶瘤病毒及其药物组合与制备和应用

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